The genomic DNA of all organisms undergoes spontaneous changes in the sequence (termed as mutation) in the course of their continuing evolution thereby generating variant forms of progenitor sequences, which may lead to various evolutionary advantages or disadvantages to the survival of the organism. If such effects of the mutations or variations are not seen then they are termed as neutral changes/mutations. If the mutation is lethal then it is not transmitted to the following generations and thus is lost from the gene pool of that organism. A variant form may also confer an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species, and hence, effectively it becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in the gene pool of the species. This coexistence of multiple forms of a sequence gives rise to polymorphisms.
Several different types of polymorphism have been reported. A restriction fragment length polymorphism (RFLP) means a variation in DNA sequence that alters the length of a restriction fragment. The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses. Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetranucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis and in a large number of genetic mapping studies. Other polymorphisms take the form of single nucleotide variations. Such polymorphisms are far more frequent than RFLPS, STRs and VNTRs. Some single nucleotide polymorphisms (SNPs) occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include beta.-globin (sickle cell anemia) and CFTR (cystic fibrosis). Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.
The effects of such polymorphisms can be at various levels of cellular organization. Polymorphic elements in the promoter and/or regulatory regions are known to modulate the levels of mRNA of the genes. Polymorphisms in the un-translated regions (UTR's) of the RNA have also been documented to regulate the transcriptional and translational rates of the genes. Their presence in the intron-exon boundaries can also lead to changes in splicing and or splice products that are formed from the native full length mRNA. Polymorphisms in the coding region may change the function of the protein if it is a non-synonymous change and if it occurs in a critical domain of the protein leading to functional changes of the protein.
Thus polymorphisms are useful in defining genomic regions (for example as genetic markers) and they may also lead to disease (for example functional polymorphisms). Numerous examples are documented in the scientific literature and persons trained in this field are familiar with it (please see Abney M et al, Am J Hum Genet 70:920-34, 2002; Baron M, Mol Psychiatry 6:143-9, 2001; Bodmer W F, Ciba Found Symp 130:215-28, 1987; Breslow J L, Physiol Rev 68:85-132, 1988; Caraballo L R and Hernandez M, Tissue Antigens 35:182-6, 1990; Levitt R C, Am J Respir Crit Care Med 150:S94-9, 1994; Xu J et al, Clin Exp Allergy 28 Suppl 5:1-5; discussion 26-8, 1998).
Atopic diseases are a clinically heterogeneous group of diseases characterized by elevated serum IgE levels and varying phenotypic expressions such as Asthma and Atopic Dermatitis (Barnes K C, Clin Exp Allergy 29 Suppl 4:47-511999; Barnes P J, Respir Res 2:64-5, 2001; Blumenthal M N and Amos D B, Chest 91:176S-184S, 1987; Thomas N S et al, Am J Respir Crit Care Med 156:S144-51, 1997). Specifically, Asthma is a chronic airway disease, affecting 15-18% of the world's population. It is mainly a childhood disorder though the age of onset can vary and is seen to be 35-45 yr. in the general population. Another case of extrinsic asthma is observed where the age of onset is above 45 yr. and is mainly due to the age induced changes in the lung function. The pathophysiology of atopic asthma is well documented. It is a T helper type 2 (Th2) mediated disorder with cytokines such as interleukin-4, interleukin-5, interleukin-13, implicated in the deviation of the immune system towards atopicity. Increased levels of these cytokines lead to elevated total serum IgE levels, eosinophil recruitment, and bronchial hyper-responsiveness that ultimately culminate in asthma pathogenesis. These interleukins are also known to interact and stimulate the alveolar cells and bronchial smooth muscle cells resulting in the clinical phenotypes of bronchial hyper-responsiveness (Barnes P J, Respir Res 2:64-5, 1999). Gene-gene and gene-environment interactions have been implicated in the development of asthma (Tay et al, Asian Pac J Allergy Immunol 17:239-42, 1999; Bleecker E R, Am J Respir Crit Care Med 156:S113-6, 1997; Cookson W, Nature, 402:B5-11, 1999).
Although inflammation is undoubtedly a cornerstone of asthma, the structural changes associated with asthma with respect to the cells and tissues of the airways have escaped the importance deserved. These structural changes are collectively given the term “Airway Remodeling” and the complex picture of asthma would be incomplete without giving this aspect its due relevance.
Various genetic studies have shown multiple loci to be associated with the disease. Asthma is therefore a multigenic disorder with a number of genes contributing minor effects leading to pathogenesis. Linkage studies, in various populations, have narrowed down the presence of susceptibility or disease genes to chromosomal locations such as 1p31, 5q31-33, 11p13, 12q13-24, 13q14, 17q12-21. However, all the causative genes and mutations have so far not been identified (Bleecker E R et al, Am J Respir Crit Care Med 156:S113-6, 1997; Blumenthal M N, Chest 91:176S-184S, 1987, Duffy D L, Epidemiol Rev 19:129-43, 1997). Recent advances in genome wide scans have identified several chromosomal regions such as 11q, 10p, 20p, 5q, 8p, 12p, 14q to be associated with asthma/atopy (Blumenthal M N et al, Hum Genet 114(2):157-164, 2004). Also a study has linked chromosomes 19, 20, 3, 12, 18, 11, 13 to mite sensitivity, which is a major risk factor for asthma (Blumenthal M N et al, Genes Immun 5(3): 226-231, 2004)
Transforming growth factor beta 1 (TGFβ1) plays an important role in airway wall remodeling, an established pathological feature in asthma (Elias et al, J Clin Invest 104:1001-1006, 1999; Redington et al, Am J Respir Crit Care Med 156:642-647, 1997). It is implicated in several aspects of fibrosis (Minshall et al, Am J Respir Cell Mol Biol 17:326-333, 1997) wherein subepithelial fibrosis is increased in severe asthmatics (Massague, Annu Rev Cell Biol 6:597-641, 1990). In addition, it decreases synthesis of enzymes that degrade the ECM (extracellular matrix), such as collagenase and stromelysin, and increases synthesis of inhibitors of these enzymes, including tissue inhibitor of metalloprotienase-1 (TIMP-1) and plasminogen activator inhibitor type-1 (PAI-1) (Massague, Annu Rev Cell Biol 6:597-641, 1990). TGFβ1 mRNA levels in eosinophils are increased in patients with severe asthma as compared to mild asthma (Minshall et al, Am J Respir Cell Mol Biol 17:326-333, 1997, Ohno et al, Am J Respir Cell Mol Biol 15:404-409, 1996). Alternatively, it prevents the development of allergic inflammation through the capacity to inhibit IgE synthesis and through inhibition of basophil and eosinophil proliferation (Taylor et al, Int Arch Allergy Immunol 135(1):73-82, 2004). Additionally, it abrogates the survival effects of hematopoietins on eosinophils and thereby induces their apoptosis (Alam et al, J Exp Med 179:1041-1045, 1.994). This complex mix of pro- and anti-inflammatory activities make TGFβ1 a promising candidate gene for asthma.
The TGFβ1 gene is located on chromosome 19q13.1-13.2 (Fujii et al, Somat Cell Mol Genet; 12:281-288) and has recently been linked to mite sensitivity (Blumenthal et al, Genes Immun 5:226-231, 2004). Studies carried out in different populations have identified various polymorphisms, such as −988C/A, −800G/A, −509C/T, 869T/C and, 915G/C. Earlier studies also reported a strong linkage disequilibrium (LD) between −509C/T, 869T/C and 915G/C (Dunning et al, Cancer Res 63(10):2610-2615, 2003; Pulleyn et al, Hum Genet. 109:623-627, 2001). Out of these SNPs, the C to T transition at −509 position has been found to be associated with elevated IgE levels (Hobbs et al, Am J Respir Crit Care Med 158:1958-1962, 1998) and TGFβ1 levels (Grainger et al, Hum Mol Genet 8:93-97, 1999). In another study, this polymorphism was associated with asthma severity (Pulleyn et al, Hum Genet 109:623-627, 2001). In the same study, four other polymorphisms, −988C/A, −800G/A, 869T/C and 915G/C were assessed for association with asthma but no significant association was found. A similar study in the Czech population showed no association with asthma (Buckova et al, Allergy 56:1236-1237, 2001).
Thus, both genetic and biochemical evidence indicate that TGFβ1 is a potential candidate gene for disease pathogenesis and/or susceptibility to disorders including asthma. To elucidate its genetic role in asthma, we have carried out a case-control study in two independent cohorts of asthma patients and controls (Nagpal et al, J Allergy Clin Immunol. 115(3):527-33, 2005). Here, we have genotyped a novel repeat (Accession number BV209662) and two SNPs, viz. −800G/A and the −509C/T, encompassing a region of 24.7 Kb and have analyzed the association of these polymorphisms independently and at the level of haplotype with asthma and also with serum TGFβ1 levels.
Moreover, there is evidence to suggest that ethnic differences exist in the susceptibility genes associated with asthma (Xu J et al, Am J Hum Genet 68:1437-46, 2001). Chromosome 19q13.1-13.2 harbors the Transforming Growth Factor Beta1 (TGFβ1) gene (consisting of 7 exons spanning a region of 23 kbp) (Fujii D et al, Somat. Cell Mol. Genet. 12:281-288, 1986). This gene mediates a complex mix of pro- and anti-inflammatory activities. Like IL-10, TGFβ1 indirectly inhibits T-cell activation by modulation of antigen presenting cell function and deactivating macrophages (Tsunawaki S et al, Nature 334:260-262, 1998). It also prevents the development of allergic inflammation through a capacity to inhibit IgE synthesis and through inhibition of mast cell proliferation. Additionally, it abrogates the survival effects of hematopoietins on eosinophils and thereby induces their apoptosis (Alam R et al, J Exp Med 179:1042-1045, 1994). It has also been shown that T cells engineered to secrete TGFβ1, in contrast to INF-γ secreting Th1 cells, could very effectively reduce airway inflammation and AHR (Hansen G et al, J Clin Invest 105:61-70). Furthermore, the blockade of TGFβ signaling in mature T cells enhanced airway inflammation and AHR, suggesting that the regulation of T cells via TGFβ reduces inflammatory responses in the lungs (Nakao A et al, J Exp Med 192:151-158). In contrast to these observations supporting an anti-inflammatory role for TGFβ1, the secretion of TGFβ1 after an allergic disorder developed contributes to fibrosis and the irreversible changes associated with airway remodeling in chronic asthma (Aubert J. D., Thorax 49:225-232, 1994). TGFβ1 is implicated in several aspects of fibrosis, including the deposition of extracellular matrix (ECM) components such as collagens type I and III, fibronectin, vitronectin, tenascin and proteoglycans (Massague J, Annu. Rev. Cell Biol. 6:597-641, 1990). In addition, it decreases synthesis of enzymes that degrade the ECM, such as collagenase and stromelysin, and increases synthesis of inhibitors of these enzymes, including tissue inhibitor of metalloprotienase-1 (TIMP-1) and plasminogen activator inhibitor type-1 (PAI-1) (Massague J, Annu. Rev. Cell Biol. 6:597-641, 1990). TGFβ1 mRNA levels in eosinophils are increased in patients with severe asthma as compared to mild asthma (Minshall et al, Am J Respir Cell Mol Biol 17:326-333, 1997; Ohno I et al, Am J Respir Cell Mol Biol 15: 404-409, 1996). Also in most, but not all studies, TGFβ1 expression correlates with basement membrane thickness and fibroblast number and/or disease severity. TGFβ1 is therefore a promising candidate gene for asthma. A PCT patent application WO0208468 of Moscowitz (2001) titled “Diagnostic polymorphisms for the TGF-beta1 promoter” identified two single nucleotide polymorphisms (SNPs) at positions 216 and 563 on the TGF-p1 Promoter and have shown association with various diseases including cancer, diabetes, COPD, coronary heart disease, hypertension, asthma, anxiety etc.
Various studies indicate that repeats are involved in gene regulation. In addition to the above mentioned SNPs, putative repetitive sequences in and around the TGFβ1 gene were identified by us using the RepeatMasker™ Software. A CT/CA repeat 24.9 kb upstream of the TGFβ1 gene was validated for distribution in our study population (Nagpal et al, J Allergy Clin Immunol. 115(3):527-33, 2005). It is biologically relevant to study repetitive sequences in and around the genes as it is a known fact that repetitive sequences found hundreds of bases away from the coding sequence may be involved in the regulation of gene expression through long-range interactions with the basic promoter and the trans-acting proteins. A change in repeat number can alter the nucleosomal positioning thereby altering transcriptional activity. Also, variable repeats can form secondary structures, which can augment or interfere with the gene expression (Hefferon et al, PNAS 101(10):3504-3509, 2004).
Thus, modulation of the levels of TGFβ1 by transcriptional and translational mechanisms may be responsible for the wide array of actions mediated by this cytokine. In light of the above evidence, it appears that TGFβ1 could be an important genetic locus affecting the predisposition of an individual towards various immunological disorders, including atopic asthma, COPD, hypertension, anxiety, arthritis, etc.
Studies carried out in different populations have identified various polymorphisms in the regulatory and coding regions of this gene. Particularly, the C to T transition at −509 position in the promoter region induces a YY1 consensus-binding site within a negative regulatory region of TGFβ1 transcription (Shrivastava et al, Nucleic Acids Research 24:5151-5155, 1994). This substitution has previously been associated with elevated IgE levels (Hobbs K et al, Am J Respir Crit Care Med 158:1958-1962, 1998) and TGFβ1 levels (Grainger et al, Hum Mol Genet 8:93-97, 1999).
In another study, this polymorphism was associated with asthma severity, with a greater proportion of individuals having the TT genotype in the severe asthmatics group as compared to the mild and control group (Pulleyn L J et al, Hum Genet 109(6):623-627; 2001). In accordance with the study in the Caucasian population (Pulleyn et al, Hum Genet 109:623-627, 2001), we have found the association of the −509C/T polymorphism with asthma and serum IgE. In our study population, the CC genotype was over-represented in the control group and on being changed to CT, a significant increase in risk was observed. We also observed that the individuals with CC genotype had the lowest TGFβ1 levels in their serum followed by CT while the TT homozygotes had the highest levels (Nagpal et al, J Allergy Clin Immunol. 115(3):527-33, 2005). Our observations support the study conducted by Silverman et al. (Am J Respir Crit Care Med 169(2):214-219, 2004). Also, the −509C/T polymorphism plays a greater role in asthma as seen in a recent study by Silverman et al (Am J Respir Crit Care Med 169(2):214-219, 2004), which showed that the C to T transition at position −509 augments Yin-Yang1 (YY1) transcription factor binding and enhances basal promoter function for TGFβ1 gene; the T allele thus being associated with higher levels of TGFβ1 transcript.
A similar study in the Czech population however showed no association with asthma (Buckova I et al, Allergy Net 1236-1237). In the same study, four other polymorphisms, −988C/A, −800G/A, 869T/C and 915G/C, were assessed for association with asthma but no significant association was found with the asthmatic phenotype.
These studies suggest that there is a component of ethnic variation that is involved which depends on the particular population under study. In the present study, the CC genotype at −509 locus is present at a higher frequency in the controls, thereby being protective in nature. The TT genotype however is present at similar frequencies in the case and control groups. Also, in initial genotyping of TGFβ1 polymorphisms, two SNPs (−988 C/A and 788 C/T), reported to be polymorphic in populations of other ethnicities were found to be non-polymorphic in the Indian population (N=200). The difference in results obtained in our population could be explained by ethnic differences that exist between our population and the other populations studied. Also, the sampling strategies used in the studies are different. The sampling strategy used by the applicants is a case control study although they have recruited individuals with a familial history of asthma and atopy.
The two SNPs assessed in the study population lie within the promoter region of the gene. Polymorphisms in the promoter region of the gene can lead to abnormal transcriptional regulation. It is known that the C to T transition at the −509 position creates a Yin-Yang1 (YY1) transcription factor binding site, which possibly augments the basal promoter function for the TGFβ1 gene (Silverman et al, Am J Respir Crit Care Med 169(2):214-219, 2004). Also the polymorphism at −800 representing a G to A transition lies in a CREB transcription factor binding site (Grainger et al, Hum Mol Genet 8:93-97, 1999).
The present invention for the first time report a novel polymorphism wherein a CT/CA repeat 24.9 kb upstream of the TGFβ1 gene has been identified. This polymorphism along with two known polymorphisms viz. G/A at −800 and C/T at −509 position in the promoter of TGFβ1 gene, has been shown to be associated with susceptibility for immunological disorders, asthma in particular. This is the first time demonstration of the association of a combination of three polymorphisms in the TGFβ1 gene with immunological disorders, particularly asthma.
Here the applicants have for the first time provided novel gene variants and haplotypes useful for prediction of immunological disorders, particularly asthma.
The invention also provides novel primers for detection of novel polymorphism, a CT/CA repeat 24.9 kb upstream of the TGFβ1 gene.
To summarize, this is the first study in any population, identifying novel protective and risk haplotypes of the TGFβ1 gene (especially in comparison to that undertaken by Silverman et al, Am J Respir Crit Care Med, 169, 214-9, 2004). These haplotypic combinations have further been functionally validated. In addition to supporting the already known association of −509C/T polymorphism with the TGFβ1 levels, it is reported here the association of the (CT)n(CA)m repeat alleles with the TGFβ1 levels. In contrast to earlier observations, where TGFβ1 was primarily implicated to mediate anti-inflammatory actions; in the present study, low TGFβ1 levels associated with protective genotypes as well as haplotypes point towards the pro-inflammatory role of this cytokine. During the development of an allergic reaction, TGFβ1 is a negative regulator of inflammation, but in the case of chronic asthma (as in the patient population studied by us), where the inflammatory conditions have already established, TGFβ1 exacerbates lung deterioration, thus having a pro-inflammatory role (Nagpal et al, J Allergy Clin Immunol 115: 527-533, 2005). Thus, both genetic and biochemical evidence indicates that TGFβ1 is a potential candidate gene for disease pathogenesis and/or susceptibility to asthma. To elucidate its role in asthma genetics, in the present study a case-control study in 2 independent cohorts namely Cohort A and Cohort B of patients with asthma and controls was performed. Here, we have genotyped a novel repeat (accession number BV209662) and 2 SNPs, −800G/A and −509C/T, encompassing a region of 24.7 kb, and have analyzed the association of these polymorphisms independently and at the level of haplotype with asthma and also with serum TGF-b1 levels.